N-terminal fusion of the N-terminal domain of bacterial enzyme I facilitates recombinant expression and purification of the human RNA demethylases FTO and Alkbh5.

ABSTRACT: Various fusion tags are commonly employed to increase the heterologous expression and solubility of aggregation-prone proteins within Escherichia coli. Herein, we present a protocol for efficient recombinant expression and purification of the human RNA demethylases Alkbh5 and FTO. Our method incorporates a novel fusion tag (the N-terminal domain of bacterial enzyme I, EIN) that dramatically increases the solubility of its fusion partner and is promptly removed upon digestion with a protease. The presented protocol allows for the production of mg amounts of Alkbh5 and FTO in 1L of both rich and minimal media. We developed a liquid chromatography-mass spectrometry (LC-MS)-based assay to confirm that both proteins are enzymatically active. Furthermore, the LC-MS method developed here is appli

SUBMITTER: Khatiwada B 

PROVIDER: S-EPMC6942671 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature